Comparative Biochemical Characterization, Purification, and Apoptosis-Mediated Anticancer Activity of Casein Proteins Isolated from Gir cattle and Jersey cattle Cow Milk Against Human Colorectal Cancer (HT-29) Cells

Comparative Biochemical Characterization, Purification, and Apoptosis-Mediated Anticancer Activity of Casein Proteins Isolated from Gir cattle and Jersey cattle Cow Milk Against Human Colorectal Cancer (HT-29) Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative Biochemical Characterization, Purification, and Apoptosis-Mediated Anticancer Activity of Casein Proteins Isolated from Gir cattle and Jersey cattle Cow Milk Against Human Colorectal Cancer (HT-29) Cells S Sivaranjani, M Archana, A Sarumathi, A Ramabhai, Rengarajan Murugesan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8343107/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Milk casein proteins are recognized for their diverse biological activities, including potential anticancer effects. Native (Gir) and Hybrid (Jersey) cow’s milk differ genetically, which may affect the composition and function of their milk proteins. Understanding these differences and their biological implications is crucial for assessing the therapeutic potential of milk-derived bioactive proteins. This study compares casein proteins extracted from Native and Hybrid cow’s milk and evaluates their cytotoxic and apoptotic effects on human colorectal cancer (HT-29) cells. Caseins were effectively isolated from both milk types through isoelectric precipitation. Biochemical analysis showed differences in protein, carbohydrate, fat, and total solids between the samples. Native-PAGE revealed three distinct protein bands in Native cow casein, whereas Hybrid cow casein showed a single band, indicating compositional variation. SDS-PAGE identified two main protein fractions, αs-casein and β-casein, in both samples. Further purification via DEAE-cellulose anion exchange chromatography produced one peak for Native casein and three for Hybrid. Gel electrophoresis confirmed protein purity with distinct bands. Cytotoxicity assays yielded IC₅₀ values of 4 mg/mL for crude Native casein and 5 mg/mL for crude Hybrid casein, while purified caseins showed much greater cytotoxicity, with IC₅₀ of 300 µg/mL. Morphological analysis of treated HT-29 cells demonstrated cell shrinkage, membrane loss, and abnormal morphology, unlike untreated cells which remained normal. DNA fragmentation indicated apoptosis. The findings reveal biochemical and functional differences between caseins from Native (Gir) and Hybrid (Jersey) milk. Native casein exhibited stronger growth inhibition of HT-29 cells than Hybrid, suggesting higher anticancer potential. The increased cytotoxicity and apoptosis induction of purified caseins underscore their promise as natural anticancer agents. Overall, Native cow casein shows greater therapeutic potential in cancer research. SDS-PAGE DEAE Native Hybrid DNA fragmentation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Cancer is a major cause of death and disability worldwide, accounting for approximately 7.6 million deaths annually. While 5–10% of cancer cases are linked to genetic factors, nearly 90% are associated with lifestyle and environmental influences such as smoking, diet, nutrition, alcohol consumption, physical inactivity, obesity, infections, and exposure to pollutants. Among these, nutrition plays a particularly important role in both cancer development and prevention. Observational studies suggest that 30–40% of all cancers could be prevented through dietary changes and modifications in eating patterns (Davoodi et al., 2013). Milk, a nutrient-dense and biologically complex fluid, is mainly water, with proteins, lactose, fat, and minerals, all of which contribute to its nutritional value (Veloso et al., 2002). It provides essential nutrients necessary for a healthy diet. Bovine milk, in particular, is a key source of proteins, lipids, amino acids, vitamins, minerals, and may also contain immunoglobulins, growth factors, hormones, cytokines, peptides, nucleotides, enzymes, and polyamines. Milk proteins are present as colloidal micelles, primarily casein micelles, which are complexes of proteins and salts, notably calcium. The composition of milk varies depending on factors such as breed, age, lactation stage, nutrition, and animal health. Certain milk proteins play roles in prime immune function, with others, like lactoferrin, serving important roles in non-immune defence. Additionally, fatty acids of various types further enhance milk’s nutritional value. The protein component of milk can be categorized into casein and whey proteins based on their solubility at pH 4.6; caseins are insoluble, whereas whey proteins remain soluble. Caseins constitute the majority, approximately 80% of total milk proteins, with whey proteins making up the remaining 20%. They form a micellar complex comprising four main subtypes: αS1-, αS2-, β-, and κ-caseins, maintained by noncovalent interactions that ensure stability in milk (Veloso et al., 2002). Milk proteins can generate bioactive peptides, consisting of 2–20 amino acid residues, during digestion or processing. Their biological activity depends on amino acid composition and sequence. Whey proteins are especially rich in branched-chain amino acids (leucine, isoleucine, valine), while casein contains higher levels of histidine, methionine, phenylalanine, and valine (Girish et al., 2016). Some domesticated cows produced A2 β-casein in their milk as early as 10,000 years ago. However, a natural mutation in European breeds introduced A1 β-casein, where proline at position 67 was replaced by histidine in the 209-amino-acid β-casein chain (Prasanta et al., 2016). This mutation created new variants of milk protein, which became prevalent in breeds such as Holstein, Jersey, and Friesian. The β-casein gene, located on chromosome 6, can exist as A1A1, A2A2, or A1A2 genotypes, resulting in corresponding milk types. Northern European breeds (e.g., Holstein, Friesian) show roughly equal frequencies of A1 and A2 alleles, while southern European breeds (e.g., Jersey) typically have about 35% A1 and 65% A2 alleles (Prasanta et al., 2016). Both casein and whey proteins act as precursors to bioactive peptides released through enzymatic proteolysis during digestion or food processing. These peptides exhibit a variety of physiological activities, including opioid-like, immunomodulatory, antihypertensive, antimicrobial, antiviral, and antioxidant effects. Recent studies have also indicated their potential anticancer properties (Galcomo Pepe et al., 2013). In vitro cell culture experiments have demonstrated that milk proteins, especially casein, offer some protective effects against cancer (Sung-Woo Park et al., 2014). Casein, which accounts for nearly 80% of bovine milk proteins, exhibits anticarcinogenic activity, particularly against colon cancer. It works by inhibiting bacterial enzymes that convert procarcinogens into carcinogens and by boosting immune function through stimulating phagocytic activity and increasing lymphocyte production (Davoodi et al., 2013). The structure of casein itself is believed to contribute to its anticarcinogenic properties. Additionally, anticancer peptides derived from casein and whey are produced during proteolysis, such as during gastrointestinal digestion or fermentation. These peptides often contain proline and lysine residues, which are linked to enhanced biological activity. Several bovine milk-derived peptides have shown anticancer potential, and epidemiological evidence suggests that higher dairy consumption is associated with a reduced incidence of bladder, breast, and colorectal cancers (Girish et al., 2016). Materials and Methods 2.1 Collection of Milk Samples Milk from native Gir and hybrid Jersey cows was collected during evening milking at Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Madhavaram Milk Colony, Chennai. The samples were promptly stored in an ice box and immediately transferred to -4°C in the laboratory. 2.2 Biochemical Analysis 2.2.1 Isolation and estimation of Casein protein Isoelectric precipitation was employed to isolate casein from pooled milk samples. Raw milk fat was separated via centrifugation at 5000 rpm for 20 minutes. The test tubes were layered with two strata: an upper fat layer and a lower casein layer. The lower layer was carefully separated, the pH was adjusted to 4.6 with 1N HCl, and the mixture was thoroughly mixed. For filtration, the precipitate was passed through double cheesecloth. The collected casein was washed with double-distilled water to remove impurities, then with ethanol to remove residual moisture. The filtrate was further washed with diethyl ether to extract excess fat. After complete washing, the casein was air-dried at room temperature and then weighed for further analysis (Santosh Kumar et al., 2013). 2.2.2 Estimation of protein The casein content in native and hybrid cow’s milk samples was quantified using the Bradford method (1976). 2.2.2 Estimation of carbohydrate The carbohydrate content of the milk samples was analysed using the Roe et al. method (1955). 2.2.3 Estimation of Fat The fat content of the samples was analysed using the Rose-Gottlieb method (1961). 2.2.4 Estimation of Total Solids The determination of total solids in the milk sample was carried out using the Gravimetric Method (1961). Analysis of crude Casein protein profile by NATIVE and SDS-PAGE 2.3.1 Native gel electrophoresis The casein protein was analysed by Native Polyacrylamide Gel Electrophoresis (Native-PAGE) as per the method of Maurer (1970). 2.3.2 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis SDS-PAGE analysis was performed on crude casein protein samples following the Laemmli (1970) method. Purification of casein protein by DEAE-Cellulose Anion Exchange Chromatography After analysing the protein profile, casein was purified using Diethylaminoethyl (DEAE) cellulose ion-exchange chromatography, according to Ivan Bonizzi et al. (2009). Preparation of DEAE – cellulose DEAE-cellulose, an anion exchanger, was obtained from Sigma-Aldrich. To swell the DEAE-cellulose, 5 g of dried matrix was soaked overnight at 10°C in 100 mL of double-distilled water. The swollen matrix was washed three times with 100 mL of double-distilled water. The suspension was then gently stirred, and 0.1 M sodium hydroxide was added, mixing 300 mL of the solution for an hour and maintaining a pH of 13. The gel was subsequently washed with double-distilled water until neutral (approximately pH 7). It was then replaced with 0.1 M HCl and stirred for 1 hour to reach pH 1, after which the HCl solution was removed. The gel was washed again with double-distilled water until the pH reached 3.0. The water was discarded, and the gel was equilibrated with 100 mM sodium phosphate buffer at pH 7.6, then degassed and fines removed. The slurry was then loaded into a Bio-Rad column. Packing the DEAE-cellulose was done gently to remove air bubbles, and the column was left undisturbed overnight. Crude casein protein (2 mL) was applied to an anion-exchange column (1.7 × 15 cm, Bio-Rad Pharma), equilibrated with 100 mM sodium phosphate buffer at pH 7.6. Protein was eluted using the same buffer and a sodium chloride gradient (0–1 M NaCl) at 1 mL/min, collecting 2 mL fractions. Protein concentrations were determined by absorbance at 280 nm. Purified protein samples were analysed by gel electrophoresis under native (Native-PAGE) and denaturing (SDS-PAGE) conditions on 12% and 15% polyacrylamide gels, respectively, following Laemmli’s (1970) method in Tris-Glycine buffer at pH 8.3. Bands were visualised with Coomassie Brilliant Blue R-250. 2.5 Characterisation of purified protein by MALDI-TOF / MS 2.5.1 Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF / MS) Tandem mass spectrometry analysis was carried out at the Australian Proteome Analysis Facility using a Q-TOF quadrupole-TOF hybrid mass spectrometer (MicroMass, Manchester, UK) equipped with a nano-spray source and borosilicate capillaries. Dried samples at room temperature were analysed with an accelerating potential of 20 kV in reflector mode. In the MALDI-TOF/MS/MS analysis, precursor ions were accelerated at 8 kV, and fragment ions from these precursors were subsequently accelerated to 19 kV. Selected spots were subjected to MS/MS analysis and MASCOT database searches. Results were BLASTed against the NCBI protein database for protein annotation and confirmation, aligning with Molle et al. (2009) for protein identity. 2.6 Anti-cancer Activity of crude and purified casein protein 2.6.1 Cancer cell line and chemicals Human colon adenocarcinoma (HT-29) and African green monkey kidney (Vero) cell lines were obtained from the National Centre for Cell Science (NCCS), Pune, India. Reagents, including Dulbecco’s Modified Eagle Medium (DMEM), Trypsin-EDTA, Fetal Bovine Serum (FBS), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Sodium Bicarbonate, DMSO, and antibiotic solutions, were purchased from Hi Media Laboratories, Mumbai, India. Cell culture supplies were obtained from Tarsons Products Pvt., Kolkata, India. 2.6.4 Toxicity assessment of crude casein protein on Vero cell line Cytotoxicity on the Vero cell line was evaluated using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) as described by Sivaranjani and Arulvasu (2018). Vero cells at a density of 5×10³ cells per millilitre were seeded in 96-well plates with DMEM medium supplemented with 10% FBS. The plates were incubated for 24 hours in a humidified atmosphere containing 5% CO₂ at 37°C. After incubation, the medium was removed, and the cells were washed with PBS. Fresh medium was added, and the mixture was incubated for 1 hour under the same conditions. The medium was then removed, and control wells received fresh medium, while treatment wells were exposed to crude casein concentrations of 1, 2, 4, 6, and 8 mg/mL. The plates were then reincubated under the same conditions. After 24 hours, 10 µL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated for an additional 3 hours. DMSO (100 µL) was added to each well to dissolve the formazan crystals, followed by gentle pipetting. Absorbance was measured at 570 nm with a microplate reader. The growth inhibition rate was then calculated. 2.6.5 Cytotoxicity of crude and purified casein protein on the HT-29 cell line by the MTT method Cell viability was assessed using the MTT assay as described by Sivaranjani and Arulvasu (2018). Human colon adenocarcinoma cells (HT-29) at a density of 5×10^3 cells/mL were seeded into 96-well plates in DMEM medium supplemented with 10% FBS. The plates were incubated in a humidified atmosphere containing 5% CO2 at 37°C for 24 hours. The medium was then removed, and the wells were washed 3 times with PBS before fresh media were added and incubation continued for an additional hour. Subsequently, the medium was aspirated, and control wells were replenished with 1 mL of medium, while treatment wells received 1–6 mg/mL of crude extract or 100–350 µg/mL of purified casein protein. The plates were incubated as before. After 24 hours, 10 µL of 5 mg/mL MTT solution was added to each well and incubated for three more hours. Then, 100 µL of DMSO was added to each well to solubilise the formazan crystals, which were mixed gently 2–3 times. The absorbance was measured at 570 nm using a microplate reader. The growth inhibition rate was then calculated: 2.6.6. Morphological observation This study investigated the effects of milk casein hydrolysates devoid of OP when administered orally in an in vivo model. A time-dependent effect on cell phenotype was observed when human colon cancer cells (HT 29) were exposed to both crude and purified casein proteins, as evidenced by morphological changes seen under inverted light microscopy. The cancer cells (5×10^4 cells/ml) were seeded in 6-well plates and cultured in DMEM medium supplemented with 10% FBS. Incubation was carried out in a 5% CO 2 humidified incubator at 37°C for 24 hours. After incubation, the medium was aspirated; control plates received fresh medium, while treatment plates were given native crude casein at IC 50 concentrations of 4 mg/ml, hybrid crude casein at 5 mg/mL, and maximum concentrations of 6 mg/ml for both native and hybrid crude casein. The IC 50 for purified native and hybrid casein was approximately 300 µg/mL, with maximum concentrations reaching 350 µg/mL. The plates were then incubated as previously described. Cells were examined under an inverted light microscope (Radical) at 40× magnification and photographed after incubation (Sivaranjani & Arulvasu, 2018). 2.6.7 Propidium iodide staining method for apoptotic nuclei Staining of apoptotic nuclei was performed following the protocol outlined by Prabhu et al. (2013). Human colon cancer cells (HT 29) at a density of 5×10^4 cells/mL were seeded into six wells containing DMEM medium supplemented with 10% FBS. The cells were incubated at 37°C in a humidified atmosphere with 5% CO 2 for 8 hours. After incubation, the medium was aspirated; control wells received fresh medium. Treatment wells were exposed to IC 50 concentrations of Native Crude Casein (ICC) at 4 mg/mL and Hybrid Crude Casein at 5 mg/mL. The maximum concentrations for Native and Hybrid crude casein are 6 mg/mL, while the IC 50 for purified Native and Hybrid casein is 300 µg/mL, with a maximum of 350 µg/mL. Following incubation, the plates were washed with PBS, fixed in a methanol: acetic acid solution (3:1 v/v) for 10 minutes, and stained with Propidium Iodide (50 µg/mL) for 20 minutes. Cells were examined under a fluorescence microscope (Radical) at 40× magnification to observe nuclear morphology immediately after staining. 2.6.8 DNA-fragmentation analysis by Agarose Gel Electrophoresis DNA extraction and agarose gel electrophoresis were performed following the method of Arulvasu et al. (2010). HT 29 (human colon cancer) cell lines at a density of 3×10^5 cells/ml were seeded into 6-well plates with DMEM medium supplemented with 10% FBS. The cell suspension was incubated for 8 hours at 37°C in a humidified incubator with 5% CO2. After incubation, the medium was removed, the plates were washed with 1X PBS, and fresh medium was added. The plates were then returned to the incubator for additional hours. Subsequently, the medium was removed again; in control wells, fresh medium was added, while in treatment wells, fresh medium was supplemented with varying concentrations of native crude casein, specifically at its IC 50 value. DNA was isolated from samples containing 150 µg/mL hygromycin, 46 pg plasmid DNA with or without competitor, native crude casein (4 mg/mL), hybrid crude casein (5 mg/mL), and the highest IC 50 concentrations of purified native and hybrid casein. Proteins were added at concentrations from 2.5 to 300 µg/mL for the lowest doses and up to 350 µg/mL for the maximum doses. After incubation as before, DNA was extracted from cell lysates. Cells were washed with PBS, lysed with 0.5 mL of lysis buffer, transferred to microfuge tubes, and incubated at 37°C for one hour. Proteinase K (4 µL) was then added, and the mixture was incubated at 50°C for three hours. To each tube, 0.5 mL of phenol-chloroform-isoamyl alcohol (25:24:1) was added, vortexed, and centrifuged at 10,000 rpm for 10 minutes to recover the DNA-containing aqueous phase. The extraction was repeated twice with phenol-chloroform and once with chloroform. DNA was precipitated by adding two volumes of ice-cold absolute ethanol and 1/10 volume of 3 M sodium acetate, then incubated on ice for 30 minutes. Precipitated DNA was pelleted by centrifugation at 13,000 rpm for 10 minutes at 4°C, supernatant discarded, and the pellet washed with 70% ethanol. The centrifugation was repeated, and the pellet was air-dried. DNA quantity was measured using UV-visible spectrophotometry. Ten micrograms of DNA was loaded onto a 1% agarose gel containing ethidium bromide in a TBE buffer system and electrophoresed at 90 V for 1 hour. The gel was visualised under UV transillumination and documented with a camera. 2.7 Statistical analysis The data obtained in the present study were subjected to standard statistical analysis using SPSS 12.5 version to calculate the standard deviations. Results 3.1. Biochemical analysis The casein protein was extracted from milk samples of Native (Gir) and Hybrid (Jersey) cows using isoelectric precipitation. The precipitated casein was then lyophilised and stored at − 20°C for future analysis. Biochemical parameters, including protein, carbohydrate, fat, and total solids, were measured for both samples using standard methods. The results are shown in Table 1 . Table 1 Biochemical composition of Native and Hybrid cow’s milk. Biochemical components Native milk sample Hybrid milk sample Protein (mg/ml) 10.7 7.6 Carbohydrate (mg/ml) 2.4 1.6 Fat (%) 4.34 5.44 Total solids (%) 16.61 16.78 3.2. Analysis of Protein Profile 3.2.1. Native–PAGE The crude casein proteins extracted from Native (Gir) and Hybrid (Jersey) cow milk samples were analysed using Native Polyacrylamide Gel Electrophoresis (Native–PAGE). A total of 4 mg of crude casein was dissolved in 1 mL of sodium phosphate buffer (pH 7.6), and 20 µL of this solution was loaded into each well along with the sample buffer. The electrophoretic pattern showed a single, prominent band in both Native and Hybrid samples, with two additional minor bands observed only in the Native casein. The major bands had similar mobility, suggesting a homologous relationship between the main proteins in both samples. The electrophoretic profiles are shown in Fig. 1 . 3.2. SDS-PAGE Electrophoretic separation of native and hybrid cow milk caseins was conducted using Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS–PAGE). The electrophoretic profiles showed two prominent bands of equal intensity and similar mobility in both casein types. The molecular weights of the crude casein proteins were estimated using a standard protein marker (BioLit, SRL; range: 14–220 kDa). The results identified two main bands within the 25–35 kDa range, along with 7–8 minor bands in both native and hybrid casein samples. These results confirm the presence of multiple protein components in the milk casein fractions. The SDS–PAGE profiles of both milk caseins are illustrated in Fig. 1 . 3.3. Purification of Casein Protein by DEAE–Cellulose Anion Exchange Chromatography Crude casein proteins from Native and Hybrid milk samples were purified using a DEAE–Cellulose (Sigma-Aldrich) anion exchange column (1.7 cm × 15 cm) packed with resin equilibrated in 0.1 M sodium phosphate buffer (pH 7.6). A crude casein (10 mg/ml) sample was applied to the column. Unbound proteins were eluted with the same buffer, while bound proteins were eluted through a 0–1 M NaCl linear gradient at a flow rate of 1 mL/min. A total of 40 fractions, including both bound and unbound proteins, were collected and monitored at 280 nm. Figures 3 and 4 display the chromatographic profiles of native and hybrid casein proteins. The native casein profile shows a single, sharp peak, indicating its homogeneous nature. Conversely, the hybrid casein profile ( Fig. 2 ) presents three distinct peaks, indicating structural heterogeneity and the presence of multiple interacting protein forms or complexes. Selected peak fractions (60 µL) were analyzed using 10% Native–PAGE and 12% SDS–PAGE. The Native–PAGE profile showed a single sharp band in fractions 29 (Native) and 24 (Hybrid), confirming the presence of purified casein. The SDS–PAGE patterns revealed two distinct bands for the Native and Hybrid casein proteins, respectively. Figure 3 illustrates the oligomeric state of the native casein protein, clearly showing a dimeric conformation based on the band patterns and structural representation. Conversely, the figure hybrid casein protein, which displays a trimeric organisation, is indicated by three associated subunits. Together, these figures confirm the different oligomeric states of native and hybrid casein proteins, emphasising the structural change in the hybrid form. 3.4. Characterisation of Purified Protein by MALDI–TOF/MS Purified casein proteins from both Native and Hybrid milk samples were analysed using MALDI–TOF mass spectrometry. The resulting spectra showed high reproducibility and clearly distinguished between the two casein types. Characteristic discriminant mass regions were identified at 353 and 2020 m/z for the Hybrid casein protein, and at 212 and 1173 m/z for the Native casein protein ( Fig. 4 , 5 ). These results validate MALDI–TOF/MS as an effective method for differentiating between Native and Hybrid milk caseins. In proteomic research, MALDI–TOF/MS has been instrumental in profiling bovine milk proteins, determining molecular weights without fractionation, and characterising glycosylation patterns in milk proteins such as milk-fat-globule membrane proteins, immunoglobulins, α-lactalbumin, κ-casein, and lactoferrin. Figure 8 MALDI – TOF analysis of purified Hybrid casein protein 3.5. Toxicity assessment of Native and Hybrid crude casein protein on Vero cell line The cytotoxicity of crude native and hybrid casein proteins was assessed using the MTT assay on African green monkey kidney (Vero) cells at concentrations of 2–8 mg/mL. The results ( Fig. 6 , 7 ) showed little to no cytotoxicity for both casein samples, indicating they are non-toxic to normal cell lines. 3.6. Anticancer Activity Against HT-29 Human Colon Cancer Cell Line The anticancer potential of crude and purified casein proteins (Native & Hybrid) was evaluated on human colon adenocarcinoma (HT-29) cells using the MTT assay. Purified casein proteins from both Native and Hybrid milk samples were characterised by MALDI–TOF mass spectrometry. The IC₅₀ values for crude Native and Hybrid caseins were 4 mg/mL and 5 mg/mL, respectively ( Fig. 8 ) , while the IC₅₀ for purified casein proteins (both Native and Hybrid) was 300 µg/mL ( Fig. 9 ). The anticancer activity is likely due to the presence of α- and β-casein, which contain bioactive peptide sequences. Notably, Native casein demonstrated greater anticancer potential than the Hybrid variant. 3.7. Cytomorphological observations Purified casein proteins from both Native and Hybrid milk samples were analysed using MALDI–TOF mass spectrometry. Control cells appeared normal with polygonal shapes and confluent monolayers. In contrast, treated cells showed significant morphological changes, including shrinkage, rounding, and loss of adhesion, indicating cytotoxic stress. At IC₅₀ concentrations (4 mg/mL and 5 mg/mL for crude Native and Hybrid caseins; 300 µg/mL for purified caseins), about 50% of cells remained viable. At higher concentrations (6 mg/mL and 350 µg/mL), marked cellular damage was observed ( Fig. 10 ). These findings suggest that casein treatment inhibits HT-29 cell proliferation, likely through contact inhibition and apoptosis induction. 3.8. Propidium Iodide (PI) Staining To confirm apoptosis, HT-29 cells were stained with propidium iodide after treatment with both crude and purified casein proteins. Treated cells showed bright red fluorescence, indicating nuclear condensation and apoptotic body formation, whereas control cells exhibited pale red staining with intact nuclei. Figure 11 depicts the effect of crude and purified caseins on PI uptake in the treated cells. A dose-dependent increase in PI-positive cells was observed after 24 hours, with higher concentrations of crude caseins (4–6 mg/mL) and purified caseins (300–350 µg/mL) resulting in a significant rise in PI staining. This steady increase in PI-positive cells confirms that casein treatment induces apoptosis-mediated cell death. 3.9. DNA Fragmentation Assay DNA fragmentation, a hallmark of apoptosis, was analysed by 1% agarose gel electrophoresis. Control cells displayed intact DNA bands, whereas treated cells showed a characteristic DNA ladder and smear pattern, indicating fragmentation. HT-29 cells treated with crude (4, 5, and 6 mg/mL) and purified (300 and 350 µg/mL) casein proteins from both Native and Hybrid sources exhibited clear evidence of apoptotic DNA cleavage. The fragmentation pattern confirmed that casein protein induces apoptosis in HT-29 cells, leading to programmed cell death characterised by chromatin condensation, cytoplasmic shrinkage, and apoptotic body formation. Discussion Cancer remains a leading cause of death worldwide. While standard treatments like chemotherapy and radiotherapy are effective, they often come with high costs and serious side effects. Additionally, many anticancer drugs can damage healthy cells, possibly leading to secondary tumours (Quita Salwa et al., 2009). This underscores the urgent need to discover natural anticancer agents that are both safe and effective. Dietary compounds with chemopreventive properties have attracted interest as promising alternatives for cancer prevention and treatment (Sah et al., 2015 ). Using nutrition-based approaches for chemoprevention offers an innovative and practical strategy to control cancer progression and enhance overall health (Nada Al-Mudallal et al., 2016). Among natural foods, milk and its proteins stand out as potential sources due to bioactive peptides with anticancer activity (Sah et al., 2015 ). Milk is nutritionally dense, providing essential nutrients such as fats, proteins, lactose, minerals, and non-fat solids, making it an important global food (Ravi Kanth et al., 2016; Elena et al., 2003). Bovine milk primarily contains two major families of proteins: caseins and whey proteins. Caseins, the dominant phosphoproteins, account for about 80% of total milk proteins (including αs2-CN, αs1-CN, β-CN, and κ-CN), while whey proteins comprise the remaining 20%. Bioactive peptides derived from caseins have important physiological functions and possess antimicrobial, antioxidant, and anticancer activities. Research indicates that casein peptides can inhibit colon cancer cell growth by interacting with somatostatin and specific opioid receptors in the intestinal tract (GalComo Pepe et al., 2013). Casein proteins from Gir and Jersey milk were extracted, purified via ion-exchange chromatography, and tested for cytotoxic effects against the human colon adenocarcinoma cell line (HT-29). The biochemical components of both milk samples were also analysed. Similar results were reported by Aduli Enoch et al. (2012), who found fat content of 4.82% and total solids of 13% in cow’s milk, consistent with earlier studies (Klemen Potocnik et al., 2011; Niero et al., 2015; Anette et al., 2016). Electrophoretic analysis (NATIVE-PAGE) of crude casein from both samples showed one major and two minor protein bands. Under denaturing conditions (SDS-PAGE), two main and several minor bands were observed—seven in native and eight in hybrid casein. The two predominant bands corresponded to αs1-CN and β-CN, with molecular weights of 25–35 kDa. These results align with previous research (Andrii Iukalo et al., 2014), which also reported similar banding patterns. The close molecular weights of αs1-CN (23,614 Da) and β-CN (23,974 Da) account for their similar migration, complicating separation. Casein purification was achieved using DEAE-cellulose anion exchange chromatography. The chromatogram displayed a single peak for native casein and three for hybrid casein. Fractions 29 (native) and 24 (hybrid) produced single protein bands on 12% NATIVE-PAGE, confirming purity. Similar purification studies have been documented in camel and dromedary milk by Si Ahmed et al. (2012) and Maryam et al. (2011). MALDI–TOF analysis indicated that the purified native casein exhibited 53% similarity to a hypothetical protein from Streptomyces daghestanicus (7,541 Da, pI 11.48), whereas hybrid casein showed 31% similarity to a hypothetical protein from Pteropus alecto (11,831 Da, pI 6.8). Both proteins exhibited comparable mass and score values, indicating similar structural properties. For cytotoxicity assessment, the MTT assay was performed on HT-29 cells at varying concentrations of crude (1–6 mg/mL) and purified (100–350 µg/mL) casein. Viable cells reduced MTT to purple formazan crystals, measured at 570 nm. The results demonstrated dose-dependent inhibition of HT-29 cell proliferation, with maximum inhibition at 6 mg/ml (crude) and 350 µg/ml (purified). The IC₅₀ values were 4 mg/ml for crude native casein, 5 mg/ml for hybrid casein, and 300 µg/mL for both purified samples. These findings are consistent with earlier studies by Mahile et al. (2017) and Nada Al-Mudallal et al. (2016), which illustrate similar dose-dependent cytotoxic effects of milk proteins across various cancer cell lines. Morphological observations under an inverted microscope showed clear differences between treated and control HT-29 cells. The treated cells exhibited typical apoptotic features, including cell shrinkage, membrane blebbing, and decreased cell density. These morphological changes aligned with previous findings by Sung-Woo Park et al. (2014), who observed similar apoptotic effects in various cancer cell lines exposed to casein and α-casein. Additionally, milk casein proteins from Gir and Jersey were extracted, purified via ion-exchange chromatography, and tested for cytotoxicity against the human colon adenocarcinoma cell line (HT-29). DNA fragmentation analysis through agarose gel electrophoresis revealed a ladder pattern in the treated samples, confirming apoptosis-induced DNA cleavage. Similar results were reported by Osama Almahdy et al. (2011) in HepG2 and HeLa cells treated with camel casein. Conclusion The study demonstrates that casein proteins isolated from Native (Gir) and Hybrid (Jersey) cow milk exhibit significant biochemical and functional differences, with Native casein showing superior anticancer activity against human colorectal cancer (HT-29) cells. Both crude and purified casein proteins induced dose-dependent cytotoxicity and apoptosis in HT-29 cells, as evidenced by morphological changes, propidium iodide staining, and DNA fragmentation assays. The enhanced cytotoxicity of purified casein proteins highlights their potential as natural anticancer agents. These findings suggest that Native cow casein, in particular, holds promise for therapeutic applications in cancer research. Further molecular and mechanistic studies are warranted to elucidate the specific pathways underlying the antiproliferative effects of casein proteins. Abbreviations A1 – A1 β-casein A2 – A2 β-casein BLAST – Basic Local Alignment Search Tool BSA – Bovine Serum Albumin CO₂ – Carbon Dioxide DAE / DEAE – Diethylaminoethyl DEAE-Cellulose – Diethylaminoethyl Cellulose DMEM – Dulbecco’s Modified Eagle Medium DNA – Deoxyribonucleic Acid DMSO – Dimethyl Sulfoxide EDTA – Ethylenediaminetetraacetic Acid FBS – Fetal Bovine Serum HT-29 – Human Colon Adenocarcinoma Cell Line IC₅₀ – Half-Maximal Inhibitory Concentration MALDI-TOF/MS – Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry MASCOT – Matrix Science Search Tool MTT – 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide m/z – Mass-to-Charge Ratio NaCl – Sodium Chloride NCCS – National Centre for Cell Science NCBI – National Center for Biotechnology Information Native-PAGE – Native Polyacrylamide Gel Electrophoresis PAGE – Polyacrylamide Gel Electrophoresis PBS – Phosphate-Buffered Saline PI – Propidium Iodide rpm – Revolutions per Minute SDS – Sodium Dodecyl Sulfate SDS-PAGE – Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis TANUVAS – Tamil Nadu Veterinary and Animal Sciences University TBE – Tris-Borate-EDTA TE Buffer – Tris-EDTA Buffer UV – Ultraviolet Vero – African Green Monkey Kidney Cell Line Declarations Author contributions CRediT: S Sivaranjani: Funding acquisition, investigation, methodology, project administration, validation, writing– original draft. M Archana: Conceptualisation, data curation, formal analysis. A Sarumathi: Conceptualisation, project administration. V Ramabhai: Conceptualisation, project administration. Rengarajan Murugesan: Formal analysis, investigation, writing– original draft, writing– review & editing. Funding There is no funding available to support this research article. Acknowledgement S Sivaranjani sincerely thanks the management of Bon Secours College for Women; R Murugesan sincerely thanks the management of Rajagiri College of Social Sciences. Ethical approval Not applicable Declaration of generative AI and AI-assisted technologies in the writing process During manuscript preparation, Grammarly was used for language editing, spelling and grammar correction, punctuation, and clarity improvement, and Turnitin was used for detecting plagiarism and AI detection. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper. Disclosure statement The authors reported no potential conflicts of interest. Data availability statement The datasets utilised and examined in this study are available from the corresponding author upon reasonable request. References Ana Veloso, C. A., Teixeira, N., Isabel Ferreira, M. P. L., & O (2002). Separation and quantification of the major casein fractions by reverse-phase high-performance liquid chromatography and urea–polyacrylamide gel electrophoresis Detection of milk adulterations. Journal of Chromatography A , 967 , 209–218. Andrii, I. V. (2014). New Approach for Isolation of Individual Caseins from Cow Milk by the Preparative Electrophoresis. Advances in Biological Chemistry , 4 , 382–387. Hang, A., Ame Hostmark, T., & Odd Harstad, M. (2007). Bovine milk in human nutrition- a review. Lipids in health and disease , 6 , 25. Boehmer, J. H., DeGrasse, J. A., McFarland, M. A., Tall, E. A., Shefcheck, K. J., Ward, J. L., & prBannerman, D. p (2010). The proteomic advantage: Label – free quantification of protein expressed in bovine milk during experimentally induced coliform mastitis. Vet Immunol Immuno pathol , 138 , 232–266. Cheema, M., Mohan, M. S., Campagna, S. R., Jurat – Fuentes, J. L., & Harte, F. M. (2015). The association of low-molecular weight hydrophobic compounds with native casein miscelles in bovine milk. Journal Of Dairy Science , 98 , 5155–5163. Clemens Kunz and Bo Lonnerdal. (1990). Human-milk proteins: analysis of casein and casein subunits by anion-exchange chromatography, gel electrophoresis, and specific staining methods. Am J C/in Nuir , 5l , 37–46. Elena Albanell, G., Caja, X., Such, M., Rovai, A. A. K., Salama, & Ramón, C. (2003). Determination of Fat, Protein, Casein, Total Solids, and Somatic Cell Count in Goat’s Milk by Near-Infrared Reflectance Spectroscopy, Journal of AOAC international., 86, NO. 4. Farah, Z., & Rüegg, M. W. (1989). The size distribution of casein micelles in camel milk. Food microstructure , 8 , 211–216. Francesco, Visioli, & Strata, A. (2014). Milk, Dairy Products, and Their Functional Effects in Humans: A Narrative Review of Recent Evidence. Adv Nutr , 5 , 131–143. Giacomo Pepe, G. C., Tenore, R., Mastrocinque, P., & Stusio (2013). and Pietro Potential Anticarcinogenic Peptides from Bovine Milk, Journal of Amino Acids, ID 939804. Hannu Korhonen. (2009). Milk derived bioactive peptides: from science to applications. Journal of functional foods , 1 , 177–187. Zucht, H. D., Raida, M., & Adermann, K., Hans-Jiirgen igert, and Wolf-Georg Forssmann (1995). Casocidin-I: a casein<zs2 activity. FEBS , 372 , 185–188. Ivan Bonizzi, J. N., & Buffoni, M. Feligini (2009). Quantification of bovine casein fractions by direct chromatographic analysis of milk. Journal of Chromatography A , 1216 , 165–168. Ivan lopez expsito, Isidra Recio. (2006). Antibacterial activity of peptides and folding variants from milk proteins. International Dairy Journal , 16 , 1294–1305. Klemen Potočnik, V., Gantner, K., Kuterovac, & Angela Cividini. (2011). Mare’s milk: composition and protein fraction in comparison with different milk species, Mare’s milk. Mljekarstvo , 61 (2), 107–113. Marie Monaco, E., Diane Bronzert, A., Douglass Tormey, C., Waalkes, P., & Lippman, M. E (1977). Casein Production by Human Breast Cancer. Cancer research , 37 , 749–754. Molle, D., Jardin, J., Piot, M., Pasco, M., Leonil, J., & Gagnaire, V. (2009). Comparison of electrospray and matrix-assisted laser desorption ionization on the same hybrid quadrupole time-of-flight tandem mass spectrometer: application to bi-dimensional lipid chromatography of proteins from bovine milk protein. J of Chrom A , 1216 , 2424–2432. Nissen, A., Bendixen, E., Ingvartsen, K. L., & Rontved, C. M. (2012). In depth analysis of low abundant proteins in bovine colostrums using different fractionation techniques. Proteomics , 12 , 2866–2878. Ryan Elias, J., Sarah Kellerby, S., & Erica Decker, A. (2008). Antioxidant Activity of Proteins and Peptides. Critical Reviews in Food Science and Nutrition , 48 , 430–441. Sah, B. N. P., Vasiljevic, T., McKechnie, S., & Donkor, O. N. (2015). Identification of Anticancer Peptides from Bovine Milk Proteins and Their Potential Roles in Management of Cancer: A Critical Review. Comprehensive Reviews in Food Science and Food Safety , 14 , 1541–4337. Santhosh kumar, Teotia, U. V. S., & Ashwani (2013). Antioxidative property of cow milk caseinates hydrolyzed with different proteases. International Journal of Pharmacy and Pharmaceutical Sciences , 5 (2), 418–422. Lee, S., & Moon Kee Chung. (2014). A milk protein, casein, as a proliferation promoting factor in prostate cancer cells, World J. mens Health., 32(2):76–82. Saadat, Y. R., Saeidi, N., Vahed, S. Z., Barzegari, A., & Barar, J. (2015). An update to DNA ladder assay for apoptosis detection, BioImpacts., 5(1), 25–28. Sivaranjani, S., & Arulvasu, C. (2018). Synthesis of silver nanoparticles using swertipunicoside and its activity on human breast cancer cell line (MCF-7). International Journal of Pharmaceutical Sciences and Research , 9 (11), 4775–4783. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8343107","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":612729947,"identity":"417c9833-d066-47e4-9fdd-51282c8e1446","order_by":0,"name":"S Sivaranjani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYBADZgYGxoYDEgY2ciDegQdEamk8YFGRZgzWkkCsTQcqzhxObAAx8Wnhn33G7HFFzR12/tnNDQdutjGnzw87/BBoi52cbgN2LRLncswNzxx7xixx52DDwZltbLkbb6cZALUkG5sdwGHNGR4zyQa2w8wMNxIbDku28eRunJ0A0nIgcRsOLfJgLf8OM8uDtPxtk0g3nJ3+Aa8WA5CWxrbDzAZALQckzhgkyEvn4LfF8AxbmWRj32FmQ7CWigTDDdI5BQcSDHD7Re4M8zbJhm+Hk+VupD/+IGHwX15+dvrmDx8q7ORweh8KkhFOBas0wK8cBOzgLPkGwqpHwSgYBaNgZAEANkdoE+KYJ1QAAAAASUVORK5CYII=","orcid":"","institution":"Bon Secours College for Women (A)","correspondingAuthor":true,"prefix":"","firstName":"S","middleName":"","lastName":"Sivaranjani","suffix":""},{"id":612729948,"identity":"886d08b3-c2de-492e-91b4-3e8b1157abc8","order_by":1,"name":"M Archana","email":"","orcid":"","institution":"University of Madras","correspondingAuthor":false,"prefix":"","firstName":"M","middleName":"","lastName":"Archana","suffix":""},{"id":612729949,"identity":"73827d83-53a7-4bd6-9d2d-7f1342d0be50","order_by":2,"name":"A Sarumathi","email":"","orcid":"","institution":"Bon Secours College for Women (A)","correspondingAuthor":false,"prefix":"","firstName":"A","middleName":"","lastName":"Sarumathi","suffix":""},{"id":612729950,"identity":"df60850c-c5d1-4738-8449-eca032aaabd3","order_by":3,"name":"A Ramabhai","email":"","orcid":"","institution":"Academy of Maritime Education and Training (AMET)","correspondingAuthor":false,"prefix":"","firstName":"A","middleName":"","lastName":"Ramabhai","suffix":""},{"id":612729951,"identity":"404301ab-e06c-4007-9972-c66c5943f31b","order_by":4,"name":"Rengarajan Murugesan","email":"","orcid":"","institution":"Rajagiri College of Social Sciences (Autonomous)","correspondingAuthor":false,"prefix":"","firstName":"Rengarajan","middleName":"","lastName":"Murugesan","suffix":""}],"badges":[],"createdAt":"2025-12-12 07:53:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8343107/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8343107/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105905423,"identity":"fe10702c-4368-43f3-9a49-7613921108f9","added_by":"auto","created_at":"2026-04-01 10:12:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":685740,"visible":true,"origin":"","legend":"\u003cp\u003ea) Qualitative analysis of crude casein protein (Lane 1: Native \u0026amp; Lane 2: Hybrid); b) Analysis of crude native and hybrid casein protein in denaturing conditions (Lane 1: Molecular maker, Lane 2: Native milk casein protein, and Lane 3: Hybrid milk casein protein).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8343107/v1/11e9eb902e4788e3fead0133.png"},{"id":105876544,"identity":"12926cfe-4c64-4d3b-85f9-9397f4ade359","added_by":"auto","created_at":"2026-04-01 05:50:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":46727,"visible":true,"origin":"","legend":"\u003cp\u003eElution pattern of ion exchange chromatography on DEAE cellulose of native cow’s casein. The column was equilibrated in 100 mM sodium phosphate buffer, pH 7.6, and eluted with a linear gradient of 0-1 M NaCl at room temperature and a flow rate of 1 mL min-1.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8343107/v1/7f1c12bfe6f3de16cc9e6f07.png"},{"id":106401791,"identity":"ba331431-61a8-401f-b49d-393ca6d5be7f","added_by":"auto","created_at":"2026-04-08 09:09:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":48195,"visible":true,"origin":"","legend":"\u003cp\u003eElution pattern of ion exchange chromatography on DEAE cellulose of Hybrid cow’s milk casein. The column was equilibrated in 100 mM sodium phosphate buffer, pH 7.6, and eluted with a linear gradient of 0-1 M NaCl at room temperature and a flow rate of 1 mL min-1.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8343107/v1/48ea51e904467aee5184da52.png"},{"id":105876549,"identity":"09907c00-5572-46c5-a291-501b186c1222","added_by":"auto","created_at":"2026-04-01 05:50:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":833216,"visible":true,"origin":"","legend":"\u003cp\u003eMALDI- TOF analysis of Purified Native casein\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8343107/v1/0f773502bca85cd459dcf93d.png"},{"id":105904766,"identity":"1fb395b7-1c56-45ae-8a4d-a98b61fbe641","added_by":"auto","created_at":"2026-04-01 10:10:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":716547,"visible":true,"origin":"","legend":"\u003cp\u003eMALDI – TOF analysis of purified Hybrid casein.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8343107/v1/722efeee6e891069e6cb42fc.png"},{"id":105904656,"identity":"9b30fc14-9062-486c-ad32-294826bc61a1","added_by":"auto","created_at":"2026-04-01 10:10:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53792,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic analysis of crude native casein treated with Vero cells at 24 hrs.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8343107/v1/5da6a086f0326e8b16bbcaa5.png"},{"id":105876546,"identity":"9d5e5774-8b1b-470b-ace3-61414075a211","added_by":"auto","created_at":"2026-04-01 05:50:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":54103,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic analysis of crude Hybrid casein treated with Vero cells at 24 hrs.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8343107/v1/545a1e3d3093852f5f33e0c3.png"},{"id":105904891,"identity":"e7de40fa-d4ea-41f2-b10a-24cd3bb98e5c","added_by":"auto","created_at":"2026-04-01 10:10:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":58342,"visible":true,"origin":"","legend":"\u003cp\u003eAnticancer activity of native cow’s crude casein against human colon cancer cell line (HT-29).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8343107/v1/eedb3e571b0608c9a8506e15.png"},{"id":105876552,"identity":"d1ae4bd8-46de-418f-9cea-8e8780dfe68b","added_by":"auto","created_at":"2026-04-01 05:50:27","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":57405,"visible":true,"origin":"","legend":"\u003cp\u003eAnticancer activity of Hybrid cow’s crude casein against human colon cancer cell line (HT-29).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8343107/v1/998adebf95d8102a26bf1642.png"},{"id":105905387,"identity":"40587887-8ba8-4b47-8746-6082e4b2ca4d","added_by":"auto","created_at":"2026-04-01 10:11:58","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":4493,"visible":true,"origin":"","legend":"\u003cp\u003eCytomorphological observation of cells treated with crude Native \u0026amp; Hybrid casein on HT-29 cells. A - IC\u003csub\u003e50\u003c/sub\u003e concentration (4 mg/ml) \u0026amp; (5 mg/ml); B - Maximum concentration (6 mg/ml) and Cytomorphological observation of cells treated with purified Native \u0026amp; Hybrid casein on HT-29 cells. A - IC\u003csub\u003e50\u003c/sub\u003e concentration (300 µg/ml); B - Maximum concentration (350 µg/ml).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-8343107/v1/5e11aa7258733f67ff9c5d2e.png"},{"id":105876550,"identity":"de508f75-f449-41c6-b7eb-1ce1e5678904","added_by":"auto","created_at":"2026-04-01 05:50:27","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":642811,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs showing the effect of Purified Hybrid casein protein on Propidium iodide-stained\u0026nbsp;HT 29\u0026nbsp;\u0026nbsp; cells (40X Magnification). A – Control; B - IC\u003csub\u003e50 \u003c/sub\u003eConcentration (300 μg/ml); C - Maximum Concentration (350 μg/ml).\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-8343107/v1/d95e7763f57c9eaeb0c5f82a.png"},{"id":106405589,"identity":"83d03f2e-123a-4b41-879f-4853f56d0096","added_by":"auto","created_at":"2026-04-08 09:27:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4450285,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8343107/v1/428c1d35-e5b6-44df-ba42-98dc99a14111.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eComparative Biochemical Characterization, Purification, and Apoptosis-Mediated Anticancer Activity of Casein Proteins Isolated from Gir cattle and Jersey cattle Cow Milk Against Human Colorectal Cancer (HT-29) Cells\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCancer is a major cause of\u0026ensp;death and disability worldwide, accounting for approximately 7.6\u0026nbsp;million deaths annually. While 5\u0026ndash;10% of cancer cases are linked to genetic factors, nearly 90% are associated with lifestyle and environmental influences such as smoking, diet, nutrition, alcohol consumption, physical inactivity, obesity, infections, and exposure to pollutants. Among these, nutrition plays a particularly important role in both cancer development and prevention. Observational studies suggest that 30\u0026ndash;40% of all cancers could be prevented through dietary changes and modifications in eating patterns (Davoodi et al., 2013). Milk, a nutrient-dense and biologically complex fluid, is mainly water, with proteins, lactose, fat, and minerals, all of which contribute to its nutritional value (Veloso et al., 2002). It provides essential nutrients necessary for a healthy diet. Bovine milk, in particular, is a key source of proteins, lipids,\u0026ensp;amino acids, vitamins, minerals, and may also contain immunoglobulins, growth factors, hormones, cytokines, peptides, nucleotides, enzymes, and polyamines. Milk proteins are present as colloidal micelles, primarily casein micelles, which are complexes of proteins and salts, notably calcium. The composition of milk varies depending on factors such as breed, age, lactation stage, nutrition, and animal health. Certain milk proteins play roles in prime immune function, with others, like lactoferrin, serving important roles in non-immune defence. Additionally, fatty acids of various types further enhance milk\u0026rsquo;s nutritional value.\u003c/p\u003e \u003cp\u003eThe protein component of milk can be categorized into casein and whey proteins based on their solubility at pH 4.6; caseins are insoluble, whereas whey proteins remain soluble. Caseins constitute the majority, approximately 80% of total milk proteins, with whey proteins making up the remaining 20%. They form a micellar complex comprising four main subtypes: αS1-, αS2-, β-, and κ-caseins, maintained by noncovalent interactions that ensure stability in milk (Veloso et al., 2002). Milk proteins can generate bioactive peptides, consisting of 2\u0026ndash;20 amino acid residues, during digestion or processing. Their biological activity depends on amino acid composition and sequence. Whey proteins are especially rich in branched-chain amino acids (leucine, isoleucine, valine), while casein contains higher levels of histidine, methionine, phenylalanine, and valine (Girish et al., 2016). Some domesticated cows produced A2 β-casein in their milk as early as 10,000 years ago. However, a natural mutation in European breeds introduced A1 β-casein, where proline at position 67 was replaced by histidine in the 209-amino-acid β-casein chain (Prasanta et al., 2016). This mutation created new variants of milk protein, which became prevalent in breeds such as Holstein, Jersey, and Friesian. The β-casein gene, located on chromosome 6, can exist as A1A1, A2A2, or A1A2 genotypes, resulting in corresponding milk types. Northern European breeds (e.g., Holstein, Friesian) show roughly equal frequencies of A1 and A2 alleles, while southern European breeds (e.g., Jersey) typically have about 35% A1 and 65% A2 alleles (Prasanta et al., 2016).\u003c/p\u003e \u003cp\u003eBoth casein and whey proteins act as precursors to bioactive peptides released through enzymatic proteolysis during digestion or food processing. These peptides exhibit a variety of physiological activities, including opioid-like, immunomodulatory, antihypertensive, antimicrobial, antiviral, and antioxidant effects. Recent studies have also indicated their potential anticancer properties (Galcomo Pepe et al., 2013). In vitro cell culture experiments have demonstrated that milk proteins, especially casein, offer some protective effects against cancer (Sung-Woo Park et al., 2014). Casein, which accounts for nearly 80% of bovine milk proteins, exhibits anticarcinogenic activity, particularly against colon cancer. It works by inhibiting bacterial enzymes that convert procarcinogens into carcinogens and by boosting immune function through stimulating phagocytic activity and increasing lymphocyte production (Davoodi et al., 2013). The structure of casein itself is believed to contribute to its anticarcinogenic properties. Additionally, anticancer peptides derived from casein and whey are produced during proteolysis, such as during gastrointestinal digestion or fermentation. These peptides often contain proline and lysine residues, which are linked to enhanced biological activity. Several bovine milk-derived peptides have shown anticancer potential, and epidemiological evidence suggests that higher dairy consumption is associated with a reduced incidence of bladder, breast, and colorectal cancers (Girish et al., 2016).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Collection of Milk Samples\u003c/h2\u003e\n \u003cp\u003eMilk from native Gir and hybrid Jersey cows was collected during evening milking at Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Madhavaram Milk Colony, Chennai. The samples were promptly stored in an ice box and immediately transferred to -4\u0026deg;C in the laboratory.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003e2.2 Biochemical Analysis\u003c/h2\u003e\n \u003cdiv id=\"Sec5\"\u003e\n \u003ch2\u003e2.2.1 Isolation and estimation of Casein protein\u003c/h2\u003e\n \u003cp\u003eIsoelectric precipitation was employed to isolate casein from pooled milk samples. Raw milk fat was separated via centrifugation at 5000 rpm for 20 minutes. The test tubes were layered with two strata: an upper fat layer and a lower casein layer. The lower layer was carefully separated, the pH was adjusted to 4.6 with 1N HCl, and the mixture was thoroughly mixed. For filtration, the precipitate was passed through\u0026ensp;double cheesecloth. The collected casein was washed with double-distilled water to remove impurities, then with ethanol to remove residual moisture. The filtrate was further washed with diethyl ether to extract excess fat. After complete washing, the casein was air-dried at room temperature and then weighed for further analysis (Santosh Kumar et al., 2013).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003e2.2.2 Estimation of protein\u003c/h2\u003e\n \u003cp\u003eThe casein content in native and hybrid cow\u0026rsquo;s milk samples was quantified using the Bradford method (1976).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003e2.2.2 Estimation of carbohydrate\u003c/h2\u003e\n \u003cp\u003eThe carbohydrate content of the milk samples was analysed using the Roe et al. method (1955).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003e2.2.3 Estimation of Fat\u003c/h2\u003e\n \u003cp\u003eThe fat content of the samples was analysed using the Rose-Gottlieb method (1961).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003e2.2.4 Estimation of Total Solids\u003c/h2\u003e\n \u003cp\u003eThe determination of total solids in the milk sample was carried out using the Gravimetric Method (1961).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of crude Casein protein profile by NATIVE and SDS-PAGE\u003c/strong\u003e\u003c/p\u003e\n\u003ch3\u003e2.3.1 Native gel electrophoresis\u003c/h3\u003e\n\u003cp\u003eThe casein protein was analysed by Native Polyacrylamide Gel Electrophoresis (Native-PAGE) as per the method of Maurer (1970).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSDS-PAGE analysis was performed on crude casein protein samples following the Laemmli (1970) method.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003ePurification of casein protein by DEAE-Cellulose Anion Exchange Chromatography\u003c/h3\u003e\n\u003cp\u003eAfter analysing the protein profile, casein was purified using Diethylaminoethyl (DEAE) cellulose ion-exchange chromatography, according to Ivan Bonizzi et al. (2009).\u003c/p\u003e\n\u003ch3\u003ePreparation of DEAE \u0026ndash; cellulose\u003c/h3\u003e\n\u003cp\u003eDEAE-cellulose, an anion exchanger, was obtained from Sigma-Aldrich. To swell the DEAE-cellulose, 5 g of dried matrix was soaked overnight at 10\u0026deg;C in 100 mL of double-distilled water. The swollen matrix was washed three times with 100 mL of double-distilled water. The suspension was then gently stirred, and 0.1 M sodium hydroxide was added, mixing 300 mL of the solution for an hour and maintaining a pH of 13. The gel was subsequently washed with double-distilled water until neutral (approximately pH 7). It was then replaced with 0.1 M HCl and stirred for 1 hour to reach pH 1, after which the HCl solution was removed. The gel was washed again with double-distilled water until the pH reached 3.0. The water was discarded, and the gel was equilibrated with 100 mM sodium phosphate buffer at pH 7.6, then degassed and fines removed. The slurry was then loaded into a Bio-Rad column. Packing the DEAE-cellulose was done gently to remove air bubbles, and the column was left undisturbed overnight. Crude casein protein (2 mL) was applied to an anion-exchange column (1.7 \u0026times; 15 cm, Bio-Rad Pharma), equilibrated with 100 mM sodium phosphate buffer at pH 7.6. Protein was eluted using the same buffer and\u0026ensp;a sodium chloride gradient (0\u0026ndash;1 M NaCl) at 1 mL/min, collecting 2 mL fractions. Protein concentrations were determined by absorbance at 280 nm. Purified protein samples were analysed by gel electrophoresis under native (Native-PAGE) and denaturing (SDS-PAGE) conditions on 12% and 15% polyacrylamide gels, respectively, following Laemmli\u0026rsquo;s (1970) method in Tris-Glycine buffer at pH 8.3. Bands were visualised with Coomassie Brilliant Blue R-250.\u003c/p\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003e2.5 Characterisation of purified protein by MALDI-TOF / MS\u003c/h2\u003e\n \u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003e2.5.1 Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF / MS)\u003c/h2\u003e\n \u003cp\u003eTandem mass spectrometry analysis was carried out at the Australian Proteome Analysis Facility using a Q-TOF quadrupole-TOF hybrid mass spectrometer (MicroMass, Manchester, UK) equipped with a nano-spray source and borosilicate capillaries. Dried samples at room temperature were analysed with an accelerating potential of 20 kV in reflector mode. In the MALDI-TOF/MS/MS analysis, precursor ions were accelerated at 8 kV, and fragment ions from these precursors were subsequently accelerated to 19 kV. Selected spots were subjected to MS/MS analysis and MASCOT database searches. Results were BLASTed against the NCBI protein database for protein annotation and confirmation, aligning with Molle et al. (2009) for protein identity.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003e2.6 Anti-cancer Activity of crude and purified casein protein\u003c/h2\u003e\n \u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003e2.6.1 Cancer cell line and chemicals\u003c/h2\u003e\n \u003cp\u003eHuman colon adenocarcinoma (HT-29) and African green monkey kidney (Vero) cell lines were obtained from the National Centre for Cell Science (NCCS), Pune, India. Reagents, including Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM), Trypsin-EDTA,\u0026ensp;Fetal Bovine Serum (FBS), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Sodium Bicarbonate, DMSO, and antibiotic solutions, were purchased from Hi Media Laboratories, Mumbai, India. Cell culture supplies were obtained from Tarsons Products Pvt., Kolkata, India.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003e2.6.4 Toxicity assessment of crude casein protein on Vero cell line\u003c/h2\u003e\n \u003cp\u003eCytotoxicity on the Vero cell line was evaluated using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) as described by Sivaranjani and Arulvasu (2018). Vero cells at a density of 5\u0026times;10\u0026sup3; cells per millilitre were seeded in 96-well plates with DMEM medium supplemented with 10% FBS. The plates were incubated for 24 hours in a humidified atmosphere containing 5% CO₂ at 37\u0026deg;C. After incubation, the medium was removed, and the cells were washed with PBS. Fresh medium was added, and the mixture was incubated for 1 hour under the same conditions. The medium was then removed, and control wells received fresh medium, while treatment wells were exposed to crude casein concentrations of 1, 2, 4, 6, and 8 mg/mL. The plates were then reincubated under the same conditions. After 24 hours, 10 \u0026micro;L of MTT solution (5 mg/mL) was added to each well, and the plates were incubated for an additional 3 hours. DMSO (100 \u0026micro;L) was added to each well to dissolve the formazan crystals, followed by gentle pipetting. Absorbance was measured at 570 nm with a microplate reader. The growth\u0026ensp;inhibition rate was then calculated.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n \u003cdiv\u003e\u003cstrong\u003e2.6.5 Cytotoxicity of crude and purified casein protein on the HT-29 cell line by the MTT method\u003c/strong\u003e\u003c/div\u003e\n \u003cp\u003eCell viability was assessed using the MTT assay as described by Sivaranjani and Arulvasu (2018). Human colon adenocarcinoma cells (HT-29) at a density of 5\u0026times;10^3 cells/mL were seeded into 96-well plates in DMEM medium supplemented with 10% FBS. The plates were incubated in a humidified atmosphere containing 5% CO2 at 37\u0026deg;C for 24 hours. The medium was then removed, and the wells were washed 3 times with PBS before fresh media were added and incubation continued for an additional hour. Subsequently, the medium was aspirated, and control wells were replenished with 1 mL of medium, while treatment wells received 1\u0026ndash;6 mg/mL of crude extract or 100\u0026ndash;350 \u0026micro;g/mL of purified casein protein. The plates were incubated as before. After 24 hours, 10 \u0026micro;L of 5 mg/mL MTT solution was added to each well and incubated for three more hours. Then, 100 \u0026micro;L of DMSO was added to each well to solubilise the formazan crystals, which were mixed gently 2\u0026ndash;3 times. The absorbance was measured at 570 nm using a microplate reader. The growth inhibition rate was then calculated:\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003e2.6.6. Morphological observation\u003c/h2\u003e\n \u003cp\u003eThis study investigated the effects of milk casein hydrolysates devoid of OP when administered orally in an in vivo model. A time-dependent effect on cell phenotype was observed when human colon cancer cells (HT 29) were exposed to both crude and purified casein proteins, as evidenced by morphological changes seen under inverted light microscopy. The cancer cells (5\u0026times;10^4 cells/ml) were seeded in 6-well plates and cultured in DMEM medium supplemented with 10% FBS. Incubation was carried out in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator at 37\u0026deg;C for 24 hours. After incubation, the medium was aspirated; control plates received fresh medium, while treatment plates were given native crude casein at IC\u003csub\u003e50\u003c/sub\u003e concentrations of 4 mg/ml, hybrid crude casein at 5 mg/mL, and maximum concentrations of 6 mg/ml for both native and hybrid crude casein. The IC\u003csub\u003e50\u003c/sub\u003e for purified native and hybrid casein was approximately 300 \u0026micro;g/mL, with maximum concentrations reaching 350 \u0026micro;g/mL. The plates were then incubated as previously described. Cells were examined under an inverted light microscope (Radical) at 40\u0026times; magnification and photographed after incubation (Sivaranjani \u0026amp; Arulvasu, 2018).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003e2.6.7 Propidium iodide staining method for apoptotic nuclei\u003c/h2\u003e\n \u003cp\u003eStaining of apoptotic nuclei was performed following the protocol outlined by Prabhu et al. (2013). Human colon cancer cells (HT 29) at a density of 5\u0026times;10^4 cells/mL were seeded into six wells containing DMEM medium supplemented with 10% FBS. The cells were incubated at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e for 8 hours. After incubation, the medium was aspirated; control wells received fresh medium. Treatment wells were exposed to IC\u003csub\u003e50\u003c/sub\u003e concentrations of Native Crude Casein (ICC) at 4 mg/mL and Hybrid Crude Casein at 5 mg/mL. The maximum concentrations for Native and Hybrid crude casein are 6 mg/mL, while the IC\u003csub\u003e50\u003c/sub\u003e for purified Native and Hybrid casein is 300 \u0026micro;g/mL, with a maximum of 350 \u0026micro;g/mL. Following incubation, the plates were washed with PBS, fixed in a methanol: acetic acid solution (3:1 v/v) for 10 minutes, and stained with Propidium Iodide (50 \u0026micro;g/mL) for 20 minutes. Cells were examined under a fluorescence microscope (Radical) at 40\u0026times; magnification to observe nuclear morphology immediately after staining.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003e2.6.8 DNA-fragmentation analysis by Agarose Gel Electrophoresis\u003c/h2\u003e\n \u003cp\u003eDNA extraction and agarose gel electrophoresis were performed following the method of Arulvasu et al. (2010). HT 29 (human colon cancer) cell lines at a density of 3\u0026times;10^5 cells/ml were seeded into 6-well plates with DMEM medium supplemented with 10% FBS. The cell suspension was incubated for 8 hours at 37\u0026deg;C in a humidified incubator with 5% CO2. After incubation, the medium was removed, the plates were washed with 1X PBS, and fresh medium was added. The plates were then returned to the incubator for additional hours. Subsequently, the medium was removed again; in control wells, fresh medium was added, while in treatment wells, fresh medium was supplemented with varying concentrations of native crude casein, specifically at its IC\u003csub\u003e50\u003c/sub\u003e value. DNA was isolated from samples containing 150 \u0026micro;g/mL hygromycin, 46 pg plasmid DNA with or without competitor, native crude casein (4 mg/mL), hybrid crude casein (5 mg/mL), and the highest IC\u003csub\u003e50\u003c/sub\u003e concentrations of\u0026ensp;purified native and hybrid casein. Proteins were added at concentrations from 2.5 to 300 \u0026micro;g/mL for the lowest doses and up to 350 \u0026micro;g/mL for the maximum doses. After incubation as before, DNA was extracted from cell lysates. Cells were washed with PBS, lysed with 0.5 mL of lysis buffer, transferred to microfuge tubes, and incubated at 37\u0026deg;C for one hour. Proteinase K (4 \u0026micro;L) was then added, and the mixture was incubated at 50\u0026deg;C for three hours. To each tube, 0.5 mL of phenol-chloroform-isoamyl alcohol (25:24:1) was added, vortexed, and centrifuged at 10,000 rpm for 10 minutes to recover the DNA-containing aqueous phase. The extraction was repeated twice with phenol-chloroform and once with chloroform. DNA was precipitated by adding two volumes of ice-cold absolute ethanol and 1/10 volume of 3 M sodium acetate, then incubated on ice for 30 minutes. Precipitated DNA was pelleted by centrifugation at 13,000 rpm for 10 minutes at 4\u0026deg;C, supernatant discarded, and the pellet washed with 70% ethanol. The centrifugation was repeated, and the pellet was air-dried. DNA quantity was measured using UV-visible spectrophotometry. Ten micrograms of DNA was loaded onto a 1% agarose gel containing ethidium bromide in a TBE buffer system and electrophoresed at 90 V for 1 hour. The gel was visualised under UV transillumination and documented with a camera.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\"\u003e\n \u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eThe data obtained in the present study were subjected to standard statistical analysis using SPSS 12.5 version to calculate the standard deviations.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Biochemical analysis\u003c/h2\u003e \u003cp\u003eThe casein protein was extracted from milk samples of Native (Gir) and Hybrid (Jersey) cows using isoelectric precipitation. The precipitated casein was then lyophilised and stored at \u0026minus;\u0026thinsp;20\u0026deg;C for future analysis. Biochemical parameters, including protein, carbohydrate, fat, and total solids, were measured for both samples using standard methods. The results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBiochemical composition of Native and Hybrid cow\u0026rsquo;s milk.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiochemical components\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNative milk sample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHybrid milk sample\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein (mg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarbohydrate (mg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFat (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal solids (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Analysis of Protein Profile\u003c/h2\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Native\u0026ndash;PAGE\u003c/h2\u003e \u003cp\u003eThe crude casein proteins extracted from Native (Gir) and Hybrid (Jersey) cow milk samples were analysed using Native Polyacrylamide Gel Electrophoresis (Native\u0026ndash;PAGE).\u003c/p\u003e \u003cp\u003eA total of 4 mg of crude casein was dissolved in 1 mL of sodium phosphate buffer (pH 7.6), and 20 \u0026micro;L of this solution was loaded into each well along with the sample buffer. The electrophoretic pattern showed a single, prominent band in both Native and Hybrid samples, with two additional minor bands observed only in the Native casein. The major bands had similar mobility, suggesting a homologous relationship between the main proteins in both samples. The electrophoretic profiles are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.2. SDS-PAGE\u003c/h2\u003e \u003cp\u003eElectrophoretic separation of native and hybrid cow milk caseins was conducted using Sodium Dodecyl Sulfate\u0026ndash;Polyacrylamide Gel Electrophoresis (SDS\u0026ndash;PAGE). The electrophoretic profiles showed two prominent bands of equal intensity and similar mobility in both casein types. The molecular weights of the crude casein proteins were estimated using a standard protein marker (BioLit, SRL; range: 14\u0026ndash;220 kDa). The results identified two main bands within the 25\u0026ndash;35 kDa range, along with 7\u0026ndash;8 minor bands in both native and hybrid casein samples. These results confirm the presence of multiple protein components in the milk casein fractions. The SDS\u0026ndash;PAGE profiles of both milk caseins are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Purification of Casein Protein by DEAE\u0026ndash;Cellulose Anion Exchange Chromatography\u003c/h2\u003e \u003cp\u003eCrude casein proteins from Native and Hybrid milk samples were purified using a DEAE\u0026ndash;Cellulose (Sigma-Aldrich) anion exchange column (1.7 cm \u0026times; 15 cm) packed with resin equilibrated in 0.1 M sodium phosphate buffer (pH 7.6). A crude casein (10 mg/ml) sample\u0026ensp;was applied to the column. Unbound proteins were eluted with the same buffer, while bound proteins were eluted through a 0\u0026ndash;1 M NaCl linear gradient at a flow rate\u0026ensp;of 1 mL/min. A total of 40 fractions, including both bound and unbound proteins, were collected and monitored at 280 nm. Figures\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e display the chromatographic profiles of native and hybrid casein proteins. The native casein profile shows a single, sharp peak, indicating its homogeneous nature. Conversely, the hybrid casein profile \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e presents three distinct peaks, indicating structural heterogeneity and the presence of multiple interacting protein forms or complexes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSelected peak fractions (60 \u0026micro;L) were analyzed using 10% Native\u0026ndash;PAGE and 12% SDS\u0026ndash;PAGE. The Native\u0026ndash;PAGE profile showed a single sharp band in fractions 29 (Native) and 24 (Hybrid), confirming the presence of purified casein. The SDS\u0026ndash;PAGE patterns revealed two distinct bands for the Native and Hybrid casein proteins, respectively. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates the oligomeric state of the native casein protein, clearly showing a dimeric conformation based on the band patterns and structural representation. Conversely, the figure hybrid casein protein, which displays a trimeric organisation, is indicated by three associated subunits. Together, these figures confirm the different oligomeric states of native and hybrid casein proteins, emphasising the structural change in the hybrid form.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Characterisation of Purified Protein by MALDI\u0026ndash;TOF/MS\u003c/h2\u003e \u003cp\u003ePurified casein proteins from both\u0026ensp;Native and Hybrid milk samples were analysed using MALDI\u0026ndash;TOF mass spectrometry. The resulting spectra showed high reproducibility and clearly distinguished between the two casein types. Characteristic discriminant mass regions were identified at 353 and 2020 m/z for the Hybrid casein protein, and at 212 and 1173 m/z for the Native casein protein \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e These results validate MALDI\u0026ndash;TOF/MS as an effective method for differentiating between Native and Hybrid milk caseins. In proteomic research, MALDI\u0026ndash;TOF/MS has been instrumental in profiling bovine milk proteins, determining molecular weights without fractionation, and characterising glycosylation patterns in milk proteins such as milk-fat-globule membrane proteins, immunoglobulins, α-lactalbumin, κ-casein, and lactoferrin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u003c/strong\u003e \u003cp\u003eMALDI \u0026ndash; TOF analysis of purified Hybrid casein protein\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Toxicity assessment of Native and Hybrid crude casein protein on Vero cell line\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of crude native and hybrid casein proteins was assessed using the MTT assay on African green monkey kidney (Vero) cells at concentrations of 2\u0026ndash;8 mg/mL. The results \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e showed little to no cytotoxicity for both casein samples, indicating they are non-toxic to normal cell lines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Anticancer Activity Against HT-29 Human Colon Cancer Cell Line\u003c/h2\u003e \u003cp\u003eThe anticancer potential of crude and purified casein proteins (Native \u0026amp; Hybrid) was evaluated on human colon adenocarcinoma (HT-29) cells using the MTT assay. Purified casein proteins from both\u0026ensp;Native and Hybrid milk samples were characterised by MALDI\u0026ndash;TOF mass spectrometry. The IC₅₀ values for crude Native and Hybrid caseins were 4 mg/mL and 5 mg/mL, respectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, while the IC₅₀ for purified casein proteins (both Native and Hybrid) was 300 \u0026micro;g/mL \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e The anticancer activity is likely due to the presence of α- and β-casein, which contain bioactive peptide sequences. Notably, Native casein demonstrated greater anticancer potential than the Hybrid variant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Cytomorphological observations\u003c/h2\u003e \u003cp\u003ePurified casein proteins from both\u0026ensp;Native and Hybrid milk samples were analysed using MALDI\u0026ndash;TOF mass spectrometry. Control cells appeared normal with polygonal shapes and confluent monolayers. In contrast, treated cells showed significant morphological changes, including shrinkage, rounding, and loss of adhesion, indicating cytotoxic stress. At IC₅₀ concentrations (4 mg/mL and 5 mg/mL for crude Native and Hybrid caseins; 300 \u0026micro;g/mL for purified caseins), about 50% of cells remained viable. At higher concentrations (6 mg/mL and 350 \u0026micro;g/mL), marked cellular damage was observed \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e These findings suggest that casein treatment inhibits HT-29 cell proliferation, likely through contact inhibition and apoptosis induction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Propidium Iodide (PI) Staining\u003c/h2\u003e \u003cp\u003eTo confirm apoptosis, HT-29 cells were stained with propidium iodide after treatment with both crude and purified casein proteins. Treated cells showed bright red fluorescence, indicating nuclear condensation and apoptotic body formation, whereas control cells exhibited pale red staining with intact nuclei. Figure\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e depicts the effect of crude and purified caseins on PI uptake in the treated cells. A dose-dependent increase in PI-positive cells was observed after 24 hours, with higher concentrations of crude caseins (4\u0026ndash;6 mg/mL) and purified caseins (300\u0026ndash;350 \u0026micro;g/mL) resulting in a significant rise in PI staining. This steady increase in PI-positive cells confirms that casein treatment induces apoptosis-mediated cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section2\"\u003e \u003ch2\u003e3.9. DNA Fragmentation Assay\u003c/h2\u003e \u003cp\u003eDNA fragmentation, a hallmark of apoptosis, was analysed by 1% agarose gel electrophoresis. Control cells displayed intact DNA bands, whereas treated cells showed a characteristic DNA ladder and smear pattern, indicating fragmentation. HT-29 cells treated with crude (4, 5, and 6 mg/mL) and purified (300 and 350 \u0026micro;g/mL) casein proteins from both Native and Hybrid sources exhibited clear evidence of apoptotic DNA cleavage. The fragmentation pattern confirmed that casein protein induces apoptosis in HT-29 cells, leading to programmed cell death characterised by chromatin condensation, cytoplasmic shrinkage, and apoptotic body formation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCancer remains a leading cause of death worldwide. While standard treatments like chemotherapy and radiotherapy are effective, they often come with high costs and serious side effects. Additionally, many anticancer drugs can damage healthy cells, possibly leading to secondary tumours (Quita Salwa et al., 2009). This underscores the urgent need to discover natural anticancer agents that are both safe and effective. Dietary compounds with chemopreventive properties have attracted interest as promising alternatives for cancer prevention and treatment (Sah et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Using nutrition-based approaches for chemoprevention offers an innovative and practical strategy to control cancer progression and enhance overall health (Nada Al-Mudallal et al., 2016). Among natural foods, milk and its proteins stand out as potential sources due to bioactive peptides with anticancer activity (Sah et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Milk is nutritionally dense, providing essential nutrients such as fats, proteins, lactose, minerals, and non-fat solids, making it an important global food (Ravi Kanth et al., 2016; Elena et al., 2003).\u003c/p\u003e \u003cp\u003eBovine milk primarily contains two major families of proteins: caseins and whey proteins. Caseins, the dominant phosphoproteins, account for about 80% of total milk proteins (including αs2-CN, αs1-CN, β-CN, and κ-CN), while whey proteins comprise the remaining 20%. Bioactive peptides derived from caseins have important physiological functions and possess antimicrobial, antioxidant, and anticancer activities. Research indicates that casein peptides can inhibit colon cancer cell growth by interacting with somatostatin and specific opioid receptors in the intestinal tract (GalComo Pepe et al., 2013). Casein proteins from Gir and\u0026ensp;Jersey milk were extracted, purified via ion-exchange chromatography, and tested for cytotoxic effects against the human colon adenocarcinoma cell line (HT-29). The biochemical components of both milk samples were also analysed. Similar results were reported by Aduli Enoch et al. (2012), who found fat content of 4.82% and total solids of 13% in cow\u0026rsquo;s milk, consistent with earlier studies (Klemen Potocnik et al., 2011; Niero et al., 2015; Anette et al., 2016).\u003c/p\u003e \u003cp\u003eElectrophoretic analysis (NATIVE-PAGE) of crude casein from both samples showed one major and two minor protein bands. Under denaturing conditions (SDS-PAGE), two main and several minor bands were observed\u0026mdash;seven in native and eight in hybrid casein. The two predominant bands corresponded to αs1-CN and β-CN, with molecular weights of 25\u0026ndash;35 kDa. These results align with previous research (Andrii Iukalo et al., 2014), which also reported similar banding patterns. The close molecular weights of αs1-CN (23,614 Da) and β-CN (23,974 Da) account for their similar migration, complicating separation. Casein purification was achieved using DEAE-cellulose anion exchange chromatography. The chromatogram displayed a single peak for native casein and three for hybrid casein. Fractions 29 (native) and 24 (hybrid) produced single protein bands on 12% NATIVE-PAGE, confirming purity. Similar purification studies have been documented in camel and dromedary milk by Si Ahmed et al. (2012) and Maryam et al. (2011).\u003c/p\u003e \u003cp\u003eMALDI\u0026ndash;TOF analysis indicated that the purified native casein exhibited 53% similarity to a hypothetical protein from \u003cem\u003eStreptomyces daghestanicus\u003c/em\u003e (7,541 Da, pI 11.48), whereas hybrid casein showed 31% similarity to a hypothetical protein from \u003cem\u003ePteropus alecto\u003c/em\u003e (11,831 Da, pI 6.8). Both proteins exhibited comparable mass and score values, indicating similar structural properties. For cytotoxicity assessment, the MTT assay was performed on HT-29 cells at varying concentrations of crude (1\u0026ndash;6 mg/mL) and purified (100\u0026ndash;350 \u0026micro;g/mL) casein. Viable cells reduced MTT to purple formazan crystals, measured at 570 nm. The results demonstrated dose-dependent inhibition of HT-29 cell proliferation, with maximum inhibition at 6 mg/ml (crude) and 350 \u0026micro;g/ml (purified). The IC₅₀ values were 4 mg/ml for crude native casein, 5 mg/ml for hybrid casein, and 300 \u0026micro;g/mL for both purified samples. These findings are consistent with earlier studies by Mahile et al. (2017) and Nada Al-Mudallal et al. (2016), which illustrate similar dose-dependent cytotoxic effects of milk proteins across various cancer cell lines.\u003c/p\u003e \u003cp\u003eMorphological observations under an inverted microscope showed clear differences between treated and control HT-29 cells. The treated cells exhibited typical apoptotic features, including cell shrinkage, membrane blebbing, and decreased cell density. These morphological changes aligned with previous findings by Sung-Woo Park et al. (2014), who observed similar apoptotic effects in various cancer cell lines exposed to casein and α-casein. Additionally, milk casein proteins from Gir and Jersey were extracted, purified via ion-exchange chromatography, and tested for cytotoxicity against the human colon adenocarcinoma cell line (HT-29). DNA fragmentation analysis through agarose gel electrophoresis revealed a ladder pattern in the treated samples, confirming apoptosis-induced DNA cleavage. Similar results were reported by Osama Almahdy et al. (2011) in HepG2 and HeLa cells treated with camel casein.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe study demonstrates that casein proteins isolated from Native (Gir) and Hybrid (Jersey) cow milk exhibit significant biochemical and functional differences, with Native casein showing superior anticancer activity against human colorectal cancer (HT-29) cells. Both crude and purified casein proteins induced dose-dependent cytotoxicity and apoptosis in HT-29 cells, as evidenced by morphological changes, propidium iodide staining, and DNA fragmentation assays. The enhanced cytotoxicity of purified casein proteins highlights their potential as natural anticancer agents. These findings suggest that Native cow casein, in particular, holds promise for therapeutic applications in cancer research. Further molecular and mechanistic studies are warranted to elucidate the specific pathways underlying the antiproliferative effects of casein proteins.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eA1 \u0026ndash; A1 \u0026beta;-casein\u003c/p\u003e\n\u003cp\u003eA2 \u0026ndash; A2 \u0026beta;-casein\u003c/p\u003e\n\u003cp\u003eBLAST \u0026ndash; Basic Local Alignment Search Tool\u003c/p\u003e\n\u003cp\u003eBSA \u0026ndash; Bovine Serum Albumin\u003c/p\u003e\n\u003cp\u003eCO₂ \u0026ndash; Carbon Dioxide\u003c/p\u003e\n\u003cp\u003eDAE / DEAE \u0026ndash; Diethylaminoethyl\u003c/p\u003e\n\u003cp\u003eDEAE-Cellulose \u0026ndash; Diethylaminoethyl Cellulose\u003c/p\u003e\n\u003cp\u003eDMEM \u0026ndash; Dulbecco\u0026rsquo;s Modified Eagle Medium\u003c/p\u003e\n\u003cp\u003eDNA \u0026ndash; Deoxyribonucleic Acid\u003c/p\u003e\n\u003cp\u003eDMSO \u0026ndash; Dimethyl Sulfoxide\u003c/p\u003e\n\u003cp\u003eEDTA \u0026ndash; Ethylenediaminetetraacetic Acid\u003c/p\u003e\n\u003cp\u003eFBS \u0026ndash; Fetal Bovine Serum\u003c/p\u003e\n\u003cp\u003eHT-29 \u0026ndash; Human Colon Adenocarcinoma Cell Line\u003c/p\u003e\n\u003cp\u003eIC₅₀ \u0026ndash; Half-Maximal Inhibitory Concentration\u003c/p\u003e\n\u003cp\u003eMALDI-TOF/MS \u0026ndash; Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry\u003c/p\u003e\n\u003cp\u003eMASCOT \u0026ndash; Matrix Science Search Tool\u003c/p\u003e\n\u003cp\u003eMTT \u0026ndash; 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide\u003c/p\u003e\n\u003cp\u003em/z \u0026ndash; Mass-to-Charge Ratio\u003c/p\u003e\n\u003cp\u003eNaCl \u0026ndash; Sodium Chloride\u003c/p\u003e\n\u003cp\u003eNCCS \u0026ndash; National Centre for Cell Science\u003c/p\u003e\n\u003cp\u003eNCBI \u0026ndash; National Center for Biotechnology Information\u003c/p\u003e\n\u003cp\u003eNative-PAGE \u0026ndash; Native Polyacrylamide Gel Electrophoresis\u003c/p\u003e\n\u003cp\u003ePAGE \u0026ndash; Polyacrylamide Gel Electrophoresis\u003c/p\u003e\n\u003cp\u003ePBS \u0026ndash; Phosphate-Buffered Saline\u003c/p\u003e\n\u003cp\u003ePI \u0026ndash; Propidium Iodide\u003c/p\u003e\n\u003cp\u003erpm \u0026ndash; Revolutions per Minute\u003c/p\u003e\n\u003cp\u003eSDS \u0026ndash; Sodium Dodecyl Sulfate\u003c/p\u003e\n\u003cp\u003eSDS-PAGE \u0026ndash; Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis\u003c/p\u003e\n\u003cp\u003eTANUVAS \u0026ndash; Tamil Nadu Veterinary and Animal Sciences University\u003c/p\u003e\n\u003cp\u003eTBE \u0026ndash; Tris-Borate-EDTA\u003c/p\u003e\n\u003cp\u003eTE Buffer \u0026ndash; Tris-EDTA Buffer\u003c/p\u003e\n\u003cp\u003eUV \u0026ndash; Ultraviolet\u003c/p\u003e\n\u003cp\u003eVero \u0026ndash; African Green Monkey Kidney Cell Line\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT:\u003c/strong\u003e \u003cstrong\u003eS Sivaranjani:\u003c/strong\u003e\u0026nbsp; Funding acquisition, investigation, methodology, project administration, validation, writing\u0026ndash; original draft.\u003cstrong\u003e\u0026nbsp;M\u003c/strong\u003e \u003cstrong\u003eArchana:\u003c/strong\u003e Conceptualisation, data curation, formal analysis. \u003cstrong\u003eA Sarumathi:\u0026nbsp;\u003c/strong\u003eConceptualisation, project administration. \u003cstrong\u003eV Ramabhai:\u003c/strong\u003e Conceptualisation, project administration. \u003cstrong\u003eRengarajan Murugesan:\u003c/strong\u003e Formal analysis, investigation, writing\u0026ndash; original draft, writing\u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no funding available to support this research article.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS Sivaranjani\u0026nbsp;\u003c/strong\u003esincerely thanks the management of Bon Secours College for Women;\u003cstrong\u003e\u0026nbsp;R Murugesan\u0026nbsp;\u003c/strong\u003esincerely thanks the management of Rajagiri College of Social Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring manuscript preparation, Grammarly was used for language editing, spelling and grammar correction, punctuation, and clarity improvement, and Turnitin was used for detecting plagiarism and AI detection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors reported no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets utilised and examined in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAna Veloso, C. 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C., Tenore, R., Mastrocinque, P., \u0026amp; Stusio (2013). and Pietro Potential Anticarcinogenic Peptides from Bovine Milk, Journal of Amino Acids, ID 939804.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHannu Korhonen. (2009). Milk derived bioactive peptides: from science to applications. \u003cem\u003eJournal of functional foods\u003c/em\u003e, \u003cem\u003e1\u003c/em\u003e, 177\u0026ndash;187.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZucht, H. D., Raida, M., \u0026amp; Adermann, K., Hans-Jiirgen igert, and Wolf-Georg Forssmann (1995). Casocidin-I: a casein\u0026lt;zs2 activity\u0026lt;=\"antibacterial=\" at=\"derived=\" exhibits=\"peptide=\"\u0026gt;\u0026lt;/zs2\u0026gt;. \u003cem\u003eFEBS\u003c/em\u003e, \u003cem\u003e372\u003c/em\u003e, 185\u0026ndash;188.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIvan Bonizzi, J. N., \u0026amp; Buffoni, M. Feligini (2009). Quantification of bovine casein fractions by direct chromatographic analysis of milk. \u003cem\u003eJournal of Chromatography A\u003c/em\u003e, \u003cem\u003e1216\u003c/em\u003e, 165\u0026ndash;168.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIvan lopez expsito, Isidra Recio. (2006). Antibacterial activity of peptides and folding variants from milk proteins. \u003cem\u003eInternational Dairy Journal\u003c/em\u003e, \u003cem\u003e16\u003c/em\u003e, 1294\u0026ndash;1305.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlemen Potočnik, V., Gantner, K., Kuterovac, \u0026amp; Angela Cividini. (2011). Mare\u0026rsquo;s milk: composition and protein fraction in comparison with different milk species, Mare\u0026rsquo;s milk. \u003cem\u003eMljekarstvo\u003c/em\u003e, \u003cem\u003e61\u003c/em\u003e(2), 107\u0026ndash;113.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarie Monaco, E., Diane Bronzert, A., Douglass Tormey, C., Waalkes, P., \u0026amp; Lippman, M. E (1977). Casein Production by Human Breast Cancer. \u003cem\u003eCancer research\u003c/em\u003e, \u003cem\u003e37\u003c/em\u003e, 749\u0026ndash;754.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMolle, D., Jardin, J., Piot, M., Pasco, M., Leonil, J., \u0026amp; Gagnaire, V. (2009). 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Antioxidative property of cow milk caseinates hydrolyzed with different proteases. \u003cem\u003eInternational Journal of Pharmacy and Pharmaceutical Sciences\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e(2), 418\u0026ndash;422.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, S., \u0026amp; Moon Kee Chung. (2014). A milk protein, casein, as a proliferation promoting factor in prostate cancer cells, World J. mens Health., 32(2):76\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaadat, Y. R., Saeidi, N., Vahed, S. Z., Barzegari, A., \u0026amp; Barar, J. (2015). An update to DNA ladder assay for apoptosis detection, BioImpacts., 5(1), 25\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSivaranjani, S., \u0026amp; Arulvasu, C. (2018). Synthesis of silver nanoparticles using swertipunicoside and its activity on human breast cancer cell line (MCF-7). \u003cem\u003eInternational Journal of Pharmaceutical Sciences and Research\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(11), 4775\u0026ndash;4783.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SDS-PAGE, DEAE, Native, Hybrid, DNA fragmentation","lastPublishedDoi":"10.21203/rs.3.rs-8343107/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8343107/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMilk casein proteins are recognized for their diverse biological activities, including potential anticancer effects. Native (Gir) and Hybrid (Jersey) cow\u0026rsquo;s milk differ genetically, which may affect the composition and function of their milk proteins. Understanding these differences and their biological implications is crucial for assessing the therapeutic potential of milk-derived bioactive proteins. This study compares casein proteins extracted from Native and Hybrid cow\u0026rsquo;s milk and evaluates their cytotoxic and apoptotic effects on human colorectal cancer (HT-29) cells. Caseins were effectively isolated from both milk types through isoelectric precipitation. Biochemical analysis showed differences in protein, carbohydrate, fat, and total solids between the samples. Native-PAGE revealed three distinct protein bands in Native cow casein, whereas Hybrid cow casein showed a single band, indicating compositional variation. SDS-PAGE identified two main protein fractions, αs-casein and β-casein, in both samples. Further purification via DEAE-cellulose anion exchange chromatography produced one peak for Native casein and three for Hybrid. Gel electrophoresis confirmed protein purity with distinct bands. Cytotoxicity assays yielded IC₅₀ values of 4 mg/mL for crude Native casein and 5 mg/mL for crude Hybrid casein, while purified caseins showed much greater cytotoxicity, with IC₅₀ of 300 \u0026micro;g/mL. Morphological analysis of treated HT-29 cells demonstrated cell shrinkage, membrane loss, and abnormal morphology, unlike untreated cells which remained normal. DNA fragmentation indicated apoptosis. The findings reveal biochemical and functional differences between caseins from Native (Gir) and Hybrid (Jersey) milk. Native casein exhibited stronger growth inhibition of HT-29 cells than Hybrid, suggesting higher anticancer potential. The increased cytotoxicity and apoptosis induction of purified caseins underscore their promise as natural anticancer agents. Overall, Native cow casein shows greater therapeutic potential in cancer research.\u003c/p\u003e","manuscriptTitle":"Comparative Biochemical Characterization, Purification, and Apoptosis-Mediated Anticancer Activity of Casein Proteins Isolated from Gir cattle and Jersey cattle Cow Milk Against Human Colorectal Cancer (HT-29) Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 05:50:18","doi":"10.21203/rs.3.rs-8343107/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fb8face7-e911-4875-9585-bc017f459a9c","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-01T05:50:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 05:50:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8343107","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8343107","identity":"rs-8343107","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}